PROJECT SUMMARY/ABSTRACT The extrinsic nervous system, enteric nerves, glia, the immune system, interstitial cells of Cajal (ICC) and smooth muscle all need to work in concert to enable coordinated motility. The Ca2+ activated Cl- channel, Ano1 is expressed in ICC and is required for ICC electrical activity. In the previous grant cycles it was shown that Ano1 regulates proliferation of ICC, that loss of Ano1 is associated with GI motility disorders, that spontaneous Ca2+ transients in the myenteric ICC network are usually coordinated but are altered and highly uncoordinated in Ano1 knockout mice, and the promoter for Ano1 was identified. How Ano1 regulates Ca2+ transients and how Ano1 is regulated is not known but what is known, though underappreciated, is that Ano1 also conducts HCO3-. The overarching novel hypothesis for this competitive renewal is that Ano1, acting with the transporter Slc4a4, regulates ICC Ca2+ transients that in turn regulate electrical activity and that transcriptional regulation of Ano1 levels in ICC by the zinc finger protein Gli physiologically regulates contractile activity. The hypothesis will be tested in two specific aims (SA). SA1 will determine how pacemaker function in myenteric ICC is regulated by pH through transport of bicarbonate ions by Ano1 and Slc4a4 (NBCe1) and SA2 will determine how transcriptional regulation of Ano1 expression by Gli contributes to normal intestinal pacemaking and GI motility. The SAs are supported by extensive preliminary data. 1) Changing extracellular HCO3- alters intracellular Ca2+ transients but not when Ano1 is blocked; 2) Pacemaker ICC differentially express the electrogenic Na+,HCO3- co-transporter Slc4a4 (NBCe1) variant c; 3) Genetic and pharmacologic block of Ano1 and Slc4a4 alter intracellular Ca2+ transients through entry of HCO3- and modulation of pH; 4) Gli1 and Gli2 bind directly to the promoter of Ano1; 5) Ano1 expression is inversely correlated with Gli levels; 6) Inhibition of Gli markedly increases Ano1 currents; 7) Upregulation of Gli alters downstream smooth muscle electrical activity, contractile patterns and GI transit; and 8) A single nucleotide variant prevents Gli binding to the Ano1 promoter and is linked to susceptibility to irritable bowel syndrome (IBS). To address the overarching hypothesis a variety of methods will be used, spanning from the use of genetically encoded indicator molecules, high speed and high resolution microscopy techniques, intracellular pH measurement, several genetically modified mouse models, electrophysiology, and improvements in the ability to obtain genetic and epigenetic sequencing information from well identified cells. Additional new expertise in genetics, transcriptional regulation, epigenetics and pH regulation has been integrated with the established team to take an innovative approach to understanding GI pacemaking in health and disease. As a result of previous work, the preliminary data, and the proposed experiments, a significant advance in the understanding of the regulation of Ano1 and how Ano1 regulates Ca2+ transients will be achieved with implications in both health and disease within and outside the GI tract.